Human circular RNA hsa_circ_0000231 clinical diagnostic effectiveness as a new tumor marker in gastric cancer

Abstract Background Owing to the subtlety of initial symptoms associated with gastric cancer (GC), the majority of patients are diagnosed at later stages. Given the absence of reliable diagnostic markers, it is imperative to identify novel markers that exhibit high sensitivity and specificity. Circular RNA, a non‐coding RNA, plays an important role in tumorigenesis and development and is well expressed in body fluids. Aims In this study, we aimed to identify hsa_circ_0000231 as a new biomarker for the diagnosis of GC and to assess its clinical diagnostic value in serum. Methods and Results The stability and correctness of hsa_circ_0000231 was determined by agarose gel electrophoresis, Rnase R assay and Sanger sequencing. Real‐time quantitative polymerase chain reaction (qRT‐PCR) was designed to discover the expression level of hsa_circ_0000231 and whether it has dynamic serum monitoring capability. The correlation between hsa_circ_0000231 and clinicopathological parameters was analyzed by collecting clinical and pathological data from GC patients. In addition, diagnostic efficacy was assessed by constructing receiver operating characteristic curves (ROC). Hsa_circ_0000231 exhibits a stable and consistently expressed structure. In GC serum, cells, and tissues, it demonstrates reduced expression levels. Elevated expression levels observed postoperatively suggest its potential for dynamic monitoring. Additionally its expression level correlates with TNM staging and neuro/vascular differentiation. The area under ROC curve (AUC) for hsa_circ_0000231 is 0.781, indicating its superior diagnostic value compared to CEA, CA19‐9, and CA72‐4. The combination of these four indicators enhances diagnostic accuracy, with an AUC of 0.833. Conclusions The stable expression of hsa_circ_0000231 in the serum of gastric cancer patients holds promise as a novel biomarker for both the diagnosis and dynamic monitoring of GC.


| INTRODUCTION
Gastric cancer (GC) is one of the most common malignant tumors in the world.The latest statistics need to show that the incidence and mortality of gastric cancer rank fifth (5.6%) and third (7.7%) in the world. 1 GC is typically discovered at an advanced stage with malignant hyperplasia and lymph node metastases due to the lack of early signs. 2 Upper gastrointestinal endoscopy and pathology biopsy remain the gold standard for clinical diagnosis, but the disadvantage is that they are overly intrusive. 3Since their introduction into the clinic, tumor markers including CEA, CA19-9, and CA72-4 have been widely employed as non-invasive liquid biopsy procedures to provide assistance in diagnosis.However, given their low sensitivity and specificity, developing effective non-invasive diagnostic markers for gastric cancer has become crucial for detection and therapy. 4,5[8] Reverse splicing creates circular RNAs (CircRNAs), singlestranded covalently closed endogenous RNA molecules that are mostly synthesized from precursor messenger RNAs lacking 5 0 end caps or 3 0 poly(A) tails. 9,10In eukaryotes, where they are extensively expressed, circRNAs were first thought to be an editing mistake with no substantial biological consequences.Scientists have recently discovered and identified hundreds of circRNAs because of improvements in genomic and transcriptomic data produced by highthroughput sequencing and bioinformatics tools. 116][17][18][19] As a result, circular RNAs have the possibility of serving as therapeutic targets and biomarkers, opening promising opportunities for the early detection, monitoring, and prediction of tumors. 20For example, Ma et al. found that hsa_circ_0004872 inhibited GC growth, invasion, and metastasis in vitro and in vivo by acting as a miR-224 "sponge" to upregulate the expression of p21 and Smad 4. 16 Their results suggest that hsa_circ_0004872 may serve as a promising diagnostic and prognostic marker for gastric cancer patients.Consequently, it is reasonable to postulate that hsa_-circ_0000231 may offer promising opportunities as a therapeutic target and biomarker for the early detection, monitoring, and prediction of tumors.
In this paper, we detected the expression level of hsa_-circ_0000231 in cells, serum and tissues of GC patients and healthy physical examination subjects, and evaluated its diagnostic efficacy in GC and its correlation with clinicopathological parameters.The changes of serum hsa_circ_0000231 in GC patients before and after operation were analyzed.

| Clinical serum and tissue specimen collection
All clinical serum and tissue samples were available from Nantong University Hospital between November 2021 and August 2022.
There were 96 patients with stomach cancer, 39 with gastritis, 26 with paired preoperative and postoperative gastric cancer patients, 74 with healthy serum samples, and 15 with paired gastric cancer and para cancerous tissue samples.In 1.5 mL RNase-free EP tubes, the top serum was withdrawn and kept as a backup at À80 C. No chemotherapy or radiation was administered before the operation for any of the patients with gastric cancer in this trial who were determined to have primary gastric cancer by qualified pathologists and physicians.

| RNA extraction
The Blood Total RNA Rapid Extraction Kit (BioTeke Corporation, Beijing, China, #RP1102) was utilized to extract total RNA from 300 μL of plasma and then used to extract RNA from serum.According to the recommendations, 600-800 mg of clinical tissue samples were extracted using TRIzol (Invitrogen, Germany, #15596018).One milliliter was lysed, extracted, and/or refrigerated at À80 C for storage.

| Real-time quantitative polymerase chain reaction
The extracted total RNA was reverse transcribed to single-stranded cDNA using a reverse transcription kit (Thermo Fisher Scientific, USA, #K1622) according to the instructions for the 20 μL system.Eleven microliter of total RNA, 4 μL of reaction buffer, 2 μL of 10 mM dNTP mix, 1 μL of random primer, 1 μL of RNase inhibitor, and 1 μL of RevertAid RT make up the kit.The combined solution was incubated in (BIO-RAD, USA) for 60 min at 42 C and 5 min at 70 C. Real-time quantitative polymerase chain reaction (qRT-PCR) experiments were performed on the acquired cDNA using a Roche Light Cycler 480 (Roche, Switzerland).Each reaction system's total reaction volume was 20 μL, which included 10 μL of SYBR Green I Mix (ABclonal, Wuhan, Chiana, #RM21203), 6 μL of enzyme-free water, 0.5 μL of primers, and 3 μL of cDNA.Ribo Biotechnology (Shanghai, China) manufactured the primers utilized in this investigation, and the target gene sequence used was hsa_circ_0000231: forward primer, 5 0 -GCTCCACTGAACAGTTTTTA-3 0 ; reverse primer, 5 0 -GGCTTGTTG GATGAATATAGCT, internal reference, 18s rRNA: reverse primer: 5 0 -CCATCCAATCGGTAGTAGCG; forward primer: 5 0 -GTAACCCGTTG AACCCAT.The relative expression of hsa_circ_0000231 was calculated by the 2 ÀΔΔCT method, ΔΔCT = mean of the experimental group (CT hsa_circ_0000231 À CT 18SrRNA ) and control group (CT hsa_circ_0000231 À CT 18SrRNA ).

| Exonuclease digestion experiments
According to the RNA extraction procedure outlined above, RNA from MKN-45 cells was extracted and divided into two equal portions.One portion of the RNA was treated with 3U Rnase R (20 U/μL, BIO-SEARCH, CA, USA, #RNR07250) at 37 C for 30 min before being inactivated with Rnase R enzyme at 70 C, while the other portion was left untreated.Rnase R, cellular RNA, 10 μL buffer, and Rnase-free water constituted a 20 μL system after reverse transcription to obtain cDNA and qRT-PCR to detect its expression level.

| RESULTS
This study aims to examine the application of hsa_circ_0000231 in the diagnosis and assessment of the efficacy of GC patients.With low expression and an expression level that relate to therapy, we discovered via a series of in vitro tests that hsa_circ_0000231 is stable and reproducible in GC and has the potential to be employed as a biomarker for GC diagnosis and efficacy observation.

| Basic information and characteristics of hsa_circ_0000231
According to the circBase database (http://www.circbase.org/),the hsa_circ_0000231 gene on chromosome 10 is the source of hsa_-circ_0000231.Exon 2 and exon 3 post-splicing make up this gene (Figure 1A).The amplification product's cyclization site was verified by Sanger sequencing to be compatible with the cyclization site reported by the CircBase database (Figure 1B).Based on the complementary DNA (cDNA) and genomic DNA (gDNA) of cells, polymerization primers and divergent primers were created.The findings revealed that hsa_circ_0000231 could only be amplified in cDNA (Figure 1C).Additionally, the loop topology of hsa_circ_0000231 prevented Rnase R from quickly degrading, while the expression level of linear hsa_circ_0000231 decreased after degradation, Figure 1D.than 5%, suggesting high accuracy (Table 1).

| Down-regulation of hsa_circ_0000231 expression in gastric cancer
The expression levels of hsa_circ_0000231 in the serum of 96 GC patients, 39 gastritis patients, and 74 healthy control patients were investigated by qRT-PCR.The results revealed that GC patients had considerably lower expression levels than healthy persons (p < .0001) and the gastritis patients' group (p < .01, Figure 2A).Next,   Hsa_circ_0000231, CEA, and CA19-9 together increased diagnostic sensitivity (97.96%),Table 3.The ability of hsa_circ_0000231 to differentiate between patients with primary gastric cancer and gastritis was equally good (AUC = 0.728, 95% CI: 0.644-0.813)(Figure 3C).

| The role of serum hsa_circ_0000231 in tumor dynamic monitoring of gastric cancer patients
To verify the dynamic relationship between plasma hsa_circ_0000231 expression and tumor progression, we compared the preoperative and postoperative paired serum hsa_circ_0000231 expression levels in 26 GC patients.The expression level of hsa_circ_0000231 was significantly lower in preoperative than in postoperative (p = .001),which offers the possibility of detecting tumor dynamics and prognosis (Figure 4A).

| Forecast hsa_circ_0000231's downstream regulatory network in GC
It was found that miRNAs can act as sponges for circRNAs involved in the construction of competitive endogenous RNAs regulating tumor progression. 21,22Nucleoplasmic separation experiments and FISH showed that hsa_circ_0000231 was slightly more abundant in the   despite being used increasingly often in recent years to treat advanced stomach cancer.Currently, invasive tissue biopsy and cytological examination are still required for the early identification of stomach cancer, but the liquid biopsy, a non-invasive tumor diagnostic method, is slowly coming into scientific view. 25,26In clinical practice, the biomarkers CEA and CA19-9 are frequently employed for adjuvant diagnosis of gastrointestinal cancers. 27Nevertheless, the early identification and prognosis of many malignancies are not restricted by the low sensitivity and specificity of these tests. 28,29Therefore, the search for effective and sensitive biomarkers is crucial.
Circular RNAs are a special class of non-coding RNAs that are structurally characterized by closed loops and do not possess conventional 5 0 and 3 0 ends. 30Due to the rapid development of highthroughput transcriptome analysis tools, thousands of circRNAs have been identified through the detection of body fluids such as plasma, serum, exosomes, and urine. 31CircRNAs, as a special class of RNA molecules, are endowed with distinctive biological functions due to their unique loop structure.The emergence of more and more data invariably reveals a striking fact: circRNAs not only play a crucial role in the onset and progression of many diseases, but also have a close association with tumor growth and proliferation. 32These research results not only highlight the great potential of circRNAs as novel biomarkers for human tumors, but also demonstrate their broad application prospects in the field of therapeutic targets.For example, hsa_circ_0007813 was up-regulated in bladder cancer, and its high expression was associated with increased tumor size, primary tumor T-stage, and pathological grading. 33Xi et al. found that CircBCAR3 promoted the proliferation, migration, and invasion of esophageal cancer cells. 34Circ_0011292 could be partially regulated through the regulation of Circ _0011292/miR-433-3p/CHEK1 axis to accelerate PTX resistance and cellular malignant progression in NSCLC cells. 35 this study, by using AGE, Sanger sequencing, and qRT-PCR in this investigation, the single-peak specificity molecule hsa_-circ_0000231 was discovered.After that, the assay was assessed for intra-and inter-batch variance.CV values were less than 5%, which showed good accuracy.7][38][39] CircRNAs can act as "competing endogenous RNAs" (ceRNAs) interacting with miRNAs to influence tumor progression.In this study, nucleoplasmic isolation and FISH assays demonstrated that hsa_circ_0000231, which is mainly distributed in the cytoplasm, can act as a miRNA sponge and participate in regulating the course of gastric cancer.

| CONCLUSION
In conclusion, our study revealed for the first time that the expression level of hsa_circ_0000231 was down-regulated in GC tissues, cells and serum and its expression level increased after gastric cancer surgery, suggesting that it can be used for dynamic monitoring of gastric cancer patients.In addition, the ROC curve also demonstrated the combined diagnostic ability of hsa_circ_0000231 with CEA, CA19-9 and CA72-4.Based on our results, hsa_circ_0000231 has the potential to be a biomarker for GC diagnosis and prognostic monitoring, but its specific function and mechanism need to be further investigated.

1. 6 g
of agarose (Biosharp, Hefei, China, #BS081-100g) was dissolved in 80 mL of 50Â TAE (Biosharp, Hefei, China, #BL533A) solution and microwaved to dissolve.Allow it cool to around 50 C, then add 10 μL of Goldview nucleic acid dye (Biosharp, Hefei, China, #BS357A), shake, and mix well before gently pouring into the electrophoresis tank along the wall and inserting the comb immediately.After the gel has formed, remove the comb, place the gel plate in the electrophoresis tank, add the electrophoresis solution, add the proper quantity of PCR product and the loading buffer to the sample wells, and set the marker wells aside as the control wells.With a gel imager, the outcomes were seen after the electrophoresis conditions were adjusted at 110 V, 40 min (BIO-RAD, USA).

GraphPad Prism 9
.0 (SanDiego, USA) was used to create the visuals, and SPSS 27.0 (IBM SPSS Statistics, Chicago, USA) was used to conduct statistical analyses of clinicopathological data.For the comparison of two independent samples, bilateral t test was used to ensure the accuracy and reliability of the results.For the comparison of multiple independent samples, ANOVA one-way analysis of variance was used to explore the differences between groups more comprehensively.In addition, in order to study the relationship between serum hsa_circ_0000231 expression and clinical pathological parameters, Chi-square test was used for statistical analysis.With p values <.05 considered as statistically significant, * means p < .05,** indicates p < .01,*** indicates p < .001,**** means p < .0001.
Figure SC,D.The mixed serum was repeatedly freeze-thawed at À80 C and room temperature for 0, 1, 3, 5, and 7 times to extract and detect the expression of hsa_circ_0000231 ( p < .0001); on the other hand, the mixed serum was left at room temperature for 0, 6, 12, 18, and 24 h to extract and detect the expression of hsa_circ_0000231 ( p < .0001),and the experiment was repeated three times, and the results showed that the expression of hsa_circ_0000231 was stable (Figure SE,F).

1
Identification of hsa_circ_0000231 loop structure.(A) Schematic diagram of hsa_circ_0000231 loci and cyclic structure in the genome.(B) The cyclization site of circ_0000231 was identified by Sanger sequencing.(C) RNase R digestion assay to confirm the stability of hsa_circ_00002311.(D) Agarose gel electrophoresis confirmed the cyclic structure of circ_0000231.*p < .05;**p < .01;**** p < .0001.T A B L E 1 The intra-assay coefficient of variation and the inter-assay coefficient of variation of hsa_Circ_0000231.

F I G U R E 3
Potential of hsa_circ_0000231 as a diagnostic biomarker.(A) receiver operating characteristic (ROC) curve analysis of plasma hsa_circ_0000231 to identify primary gastric cancer (GC) patients from healthy donors (AUC = 0.781).(B) ROC curve analysis of plasma hsa_circ_0000231 for the identification of patients with primary gastric cancer and patients with gastritis (AUC = 0.728).(C) CEA: AUC = 0.684, CA19-9: AUC = 0.627, CA72-4: AUC = 0.595 and combined diagnostic efficacy: AUC = 0.833.F I G U R E 4 Dynamic monitoring of hsa_circ_0000231 and prediction of downstream regulatory network.(A) Changes in hsa_circ_0000231 expression before and after treatment (p = .001,n = 26).(B) Nuclear plasma isolation assay to determine the binding site of hsa_circ_0000231 in GC cell line MKN-45.(C) Fluorescence in situ hybridization (FISH) analysis of the cellular distribution of hsa_circ_0000231 and 18s.(D) Bioinformatics analysis was used to predict and screen possible miRNA candidates.(E) miRNA with expression levels in gastric cancer predicted by starbase.***p< .001.